The central role of protein phosphorylation and dephosphorylation in the action of a variety of hormones is well established. A cytoplasmic cyclic AMP dependent protein kinase mediates the phosphorylation of several critical enzymes in glycogen and triglyceride metabolism, and represents a major effector system for hormones which stimulate adenylate cyclase, such as epinephrine and glucagon. Protein phosphatases also may be under hormonal control via cyclic AMP. At present, however, there remain large gaps in our understanding of overall phosphoprotein metabolism. The number and subcellular distribution of protein kinases and phosphatase is largely unknown. The spectrum of endogenous substrates is unclear and the impact of insulin on phosphoprotein metabolism has not been fully elucidated. The proposed studies are directed at obtaining an integrated view of phosphoprotein metabolism in adipocytes, as well as its hormonal control. Comparative studies of P32 phosphopeptide formation in intact fat cells as well as in subcellular fractions (plasma membrane, mitochondria, endoplasmic reticulum, nuclei and cytoplasma) have been undertaken. P32 phosphopeptides are quantified by scintillation counting and radioautography, after analytic peptide separation by polyacrylamide gel electrophoresis in detergent. These experiments will define the naturally occurring phosphopeptides as well as the variety and distribution of protein kinases and phosphatases. The qualitative and quantitative pattern of phosphopeptides formed in response to various hormones will provide a "fingerprint" of that hormone's effect, which may serve as an assay for new "second messengers". The relationship of membrane protein phosphorylation to the control of glucose transport, and adenylate cyclase activity in the plasma membrane, as well as pyruvate dehydrogenase activity in the mitochondria, will be investigated.